Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity.
Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity.
复制标题
DOI：
10.1093/nar/gkj426
复制
发表时间：
2006
影响因子：
14.9
通讯作者：
Cristofaro JV
中科院分区：
文献类型：
--
作者：
DeStefano JJ;Cristofaro JV
发展了一种基于SELEX(通过指数富集法的配体的系统进化)的方法来确定HIV-RT是否表现出对特定的引物-模板序列的偏好。设计了70个核苷酸的双链DNA，在3‘和5’端有20个核苷酸的固定侧翼序列，以及随机的30个核苷酸的内部序列。5‘端的固定序列包含从随机化区域移去6个碱基的BbsI位点。BbsI在其识别位点的下游切割，产生四个带有凹陷的3‘末端的碱基5’突起。裂解产生50个核苷酸的模板和46个核苷酸的引物，3‘末端在随机区域内。将HIV-RT与该底物孵育，通过凝胶移位分离与RT结合的物质。回收的材料经处理再生BbsI酶切位点，经聚合酶链式反应(PCR)扩增，BbsI酶切，HIV-RT筛选。这一过程重复了12轮。来自第12轮的材料比最初的材料结合得更紧密约10倍。所有选定的第12轮引物模板都具有相似的序列构型，在3‘端具有6-8个碱基G，类似于HIV多聚尿路。进一步的修饰表明，Gs对于强结合是必要的和充分的。
A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3′ and 5′ ends and a randomized 30 nt internal sequence. The fixed sequence at the 5′ end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5′ overhangs with recessed 3′ termini. Cleavage produced a 50 nt template and 46 nt primer with the 3′ terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6–8 base G run at the 3′ primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding.
登录
查看更多内容
影响因子：
2.9
作者：
CHEN, H;GOLD, L
通讯作者：
GOLD, L
影响因子：
56.9
作者：
PELISKA, JA;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ
影响因子：
3.5
作者：
JOYCE, GF
通讯作者：
JOYCE, GF
影响因子：
4.8
作者：
DeStefano, JJ;Cristofaro, JV;Fitzgerald-Heath, MJ
通讯作者：
Fitzgerald-Heath, MJ
DOI：
10.1073/pnas.90.13.6320
发表时间：
1993-07-01
影响因子：
11.1
作者：
JACOBOMOLINA, A;DING, JP;ARNOLD, E
通讯作者：
ARNOLD, E